The invention is in the field of spectral calibration of fluorescence based automated polynucleotide length measurement instruments.
Spectral calibration is to estimate reference spectral profiles (reference spectra) of particular fluorescent dyes using the optical measurement system of an automated DNA sequencer or similar fluorescent polynucleotide separation apparatus where the particular dyes will be utilized. The current practice of spectral calibration relies on measuring the spectral profile of each fluorescent dye separately. This approach to spectral calibration of fluorescent polynucleotide separation apparatus results in reduced throughput because it requires N lanes on gel-based instruments and requires N separate runs on capillary-based instrument. As more fluorescent dyes are developed and utilized routinely (N is expected to increase), the spectral calibration of fluorescent polynucleotide separation apparatus becomes more demanding and less efficient under the current practice. Additionally, the amount of computer resources devoted to spectral calibration also increases with the number of dyes and separation channels analyzed.
The invention relates to methods, compositions, and systems for calibrating a fluorescent polynucleotide separation apparatus. Fluorescent polynucleotide separation apparatus, such as an automated DNA sequencer, must be spectrally calibrated for use with the different fluorescent dyes to be used in conjunction with the separation system.
One aspect of the invention is multiple color calibration standards and their use. A multiple color calibration standard is a mixture of at least two polynucleotide of different length, wherein each of the polynucleotide is labeled with a spectrally distinct fluorescent dye. In a preferred embodiment of the invention, the multiple color calibration standard comprises at least four polynucleotides of different length, and each of the polynucleotides is labeled with a spectrally distinct dye. The invention includes numerous methods of spectrally calibrating a fluorescent polynucleotide separation apparatus with a multiple color calibration standard.
Another aspect of the invention is to produce total emission temporal profiles of multiple color calibration standards for use in calibrating fluorescent polynucleotide separation apparatus. A total emission temporal profile is a sum of the intensities of the fluorescence signal obtained in all spectral channels as a function of time. The peaks corresponding to the fluorescently labeled polynucleotides in the total emission temporal profile may be detected using a peak detector that is driven by changes in the slopes of the total emission temporal profile. Calibration of fluorescent polynucleotide separation apparatus, with various embodiments of the methods of the invention, includes the step of identification of the labeled polynucleotides of the multiple color calibration standards. The process of spectral calibration of fluorescent polynucleotide separation apparatus using a multiple color calibration standard may include the step of the estimating (extracting) of the dyes"" reference spectra, using information from the peak detection process performed on the total emission temporal profile.
Other aspects of the invention include systems for separating and detecting fluorescently labeled polynucleotides, wherein the system is designed for spectral calibration in accordance with the subject calibration methods employing multiple color calibration standards.
Other aspects of the invention include systems for separating and detecting fluorescently labeled polynucleotide, wherein the system is designed for spectral calibration in accordance with the subject calibration methods employing multiple color calibration standards. The subject systems comprise a fluorescent polynucleotide separation apparatus and a computer in functional combination with the apparatus.
Another aspect of the invention is methods and compositions for detecting the flow of electrical current through a separation channel of a fluorescent polynucleotide separation apparatus. These methods and compositions employ monitoring dyes. Monitoring dyes are fluorescent dyes that are spectrally distinct from the dye on the polynucleotide intended to convey genetic information, e.g., fluorescent polynucleotide sequencing reaction products.